1. Field of the Invention
This invention relates to a method for the rapid detection and quantification of butyric acid-producing bacteria using real-time quantitative polymerase chain reactions (qPCR).
2. Description of Related Art
Microbiologically influenced corrosion is a significant problem affecting the gas and oil industry as well as other industries, and the monitoring of microbial populations within such industries is a component of overall corrosion control operations. Traditionally, the monitoring of microbial populations in samples obtained from gas and oil production operations has employed microbial growth tests. In such tests, samples are diluted to various levels and used to inoculate microbial growth media that is designed to favor the growth of various types of bacteria. After days to several weeks of incubation, the growth tests are scored based on the presence or absence of growth in these various microbiological media. Unfortunately, as numerous researches show, only about 0.1% to about 10% bacteria from environmental samples can actually grow in an artificial medium, and a significant portion of bacteria growing in the media are not actually the target bacteria. Therefore, growth tests are unable to provide the accurate quantification of target bacteria in the samples.
To circumvent problems associated with such growth-based methods, many culture-independent genetic techniques have been developed in the past decade and applied for the detection of pathogens and quality control and assurance of products in the medicine and food industries. Because many ecosystems, including pipeline systems, have a relatively low abundance of microorganisms, the polymerase chain reaction (PCR) has been widely used to amplify the genetic signals of microbes in complex environmental samples. However, traditional PCR-based methods are significantly biased by amplification efficiency and the depletion of PCR reagents. Real-time quantitative PCR (qPCR) has been developed and used in the last few years in the medical and food research/industries to detect and quantify a number of pathogenic or infectious microorganisms. Quantitative PCR has also been used to determine the abundance of microorganisms in many different types of complex environmental samples such as sediments, water, wastewater, and marine samples. The advantage of quantitative PCR over traditional PCR is that it provides more accurate and reproducible quantification of microorganisms because quantitative PCR quantifies PCR products during the logarithmic phase of the reactions. Moreover, quantitative PCR offers a dynamic detection range of six orders of magnitude or more, does not need post-PCR manipulation, and has the capability of high throughput analysis.
Acid-producing bacteria are present in a variety of environments, including oil- and gas-bearing formations, soils, and domestic, industrial, and mining wastewaters. Acid-producing bacteria produce organic acids such as acetic, butyric, formatic, lactic, succinic, and propionic acids, and contribute to metal corrosion.
While the total bacterial number in an oil or gas production sample indicates the general conditions for bacteria to grow in this unique environment, and, thus, the potential risk of microbial corrosion, some specific groups of microorganisms commonly found in such samples probably play a more important role in the corrosion process. One such microorganism is a butyric acid-producing bacteria which has been found to be present in environmental samples and, in particular, samples from gas and oil production operations.